Published May 4, 2017 JCB: Article

WASP and SCAR are evolutionarily conserved in -filled pseudopod-based motility

Lillian K. Fritz-Laylin, Samuel J. Lord, and R. Dyche Mullins

Department of Cellular and Molecular Pharmacology, Howard Hughes Medical Institute, University of California, San Francisco, San Francisco, CA 94143

Diverse eukaryotic cells crawl through complex environments using distinct modes of migration. To understand the un- derlying mechanisms and their evolutionary relationships, we must define each mode and identify its phenotypic and molecular markers. In this study, we focus on a widely dispersed migration mode characterized by dynamic actin-filled pseudopods that we call “α-motility.” Mining genomic data reveals a clear trend: only organisms with both WASP and SCAR/WAVE—activators of branched actin assembly—make actin-filled pseudopods. Although SCAR has been shown to drive pseudopod formation, WASP’s role in this process is controversial. We hypothesize that these genes collectively represent a genetic signature of α-motility because both are used for pseudopod formation. WASP depletion from human neutrophils confirms that both are involved in explosive actin polymerization, pseudopod formation, and cell migration. WASP and WAVE also colocalize to dynamic signaling structures. Moreover, retention of WASP together

with SCAR correctly predicts α-motility in disease-causing chytrid fungi, which we show crawl at >30 µm/min with Downloaded from actin-filled pseudopods. By focusing on one migration mode in many eukaryotes, we identify a genetic marker of pseu- dopod formation, the morphological feature of α-motility, providing evidence for a widely distributed mode of cell crawling with a single evolutionary origin.

Introduction on June 21, 2017

Eukaryotic cells move using several distinct modes of locomo- leading-edge membrane, but in these cells, the force-generating tion, including crawling and fagella-driven swimming. The ste- polymer networks are composed of major rather reotyped architecture of fagella and the conservation of their than actin (Rodriguez et al., 2005). Given this variety of crawling protein components make the evolutionary conservation of cell behaviors, it is clear that one cannot simply assume that the under- swimming clear. In contrast, “crawling motility” is a collection of lying molecular mechanisms are the same. distinct processes whose evolutionary relationships are not well The best-understood mode of crawling is the slow (1–10 understood (Rodriguez et al., 2005; Lämmermann and Sixt, 2009; µm/h) creeping of adherent animal cells, including fbroblasts Paluch and Raz, 2013). Some crawling cells require dedicated and epithelial cells (Petrie and Yamada, 2015). These cells adhesion molecules to make specifc, high-affnity contacts with move by extending across a surface a sheet-like protrusion their surroundings, whereas other cells rely on weaker, nonspe- called a lamellipodium while also gripping substrate molecules THE JOURNAL OF CELL BIOLOGY CELL OF JOURNAL THE cifc interactions. Crawling cells also use different mechanisms to using integrins, which are often clustered into large focal adhe- advance their leading edge, either assembling polymerized actin sions. Although clinically and physiologically important, this networks to push the plasma membrane forward or detaching the form of adhesion-based crawling is unique to the animal lin- membrane from the underlying to form a rapidly ex- eage and is largely restricted to molecular highways formed by panding bleb. Furthermore, some cell types have been shown to use the extracellular matrix. contractile forces to generate forward movement (Lämmermann In contrast, many motile cells—including free-living et al., 2008; Bergert et al., 2012; Liu et al., 2015). Different cells amoebae and human immune cells—make 3D actin-flled pseu- can also use different sets of molecules to drive similar modes of dopods and navigate complex environments at speeds exceed- crawling. In an extreme example, sperm have evolved ing 20 µm/min (100–1,000× faster than fbroblasts) without a method of crawling in which polymer assembly advances the forming specifc molecular adhesions (Buenemann et al., 2010; Butler et al., 2010). Although this mode of fast cell crawling Correspondence to Lillian K. Fritz-Laylin: [email protected]; or R. Dyche has been called “ameboid motility,” this term is also used to Mullins: [email protected] describe a range of behaviors, including cell motility that relies L.K. Fritz-Laylin’s present address is Dept. of Biology, University of Massachu- on membrane blebs rather than actin-flled pseudopods (Läm- setts, Amherst, MA 01003. mermann and Sixt, 2009). Abbreviations used: Bd, Batrachochytrium dendrobatidis; fMLP, fMet-Leu-Phe; KD, knockdown; N-WASP, neural WASP; SCAR, suppressor of cAMP recep- tor; TIRF, total internal reflection fluorescence; WASP, Wiskott-Aldrich syndrome © 2017 Fritz-Laylin et al. This article is available under a Creative Commons License (Attribution protein; WAVE, WASP family verprolin-homologous protein. 4.0 International, as described at https ://creativecommons .org /licenses /by /4 .0 /). Supplemental Material can be found at: /content/suppl/2017/05/03/jcb.201701074.DC1.html The Rockefeller University Press $30.00 J. Cell Biol. Vol. 216 No. 6 1673–1688 https://doi.org/10.1083/jcb.201701074 JCB 1673 Published May 4, 2017

To narrow our focus, we use the term “α-motility” specif- neither family is found in all eukaryotic lineages, making them cally to describe cell crawling that is characterized by: (i) highly appealing candidates for genetic markers of α-motility. dynamic 3D pseudopods at the leading edge that are flled with SCAR is generally accepted to play a major role in the branched actin networks assembled by the Arp2/3 complex; formation of protrusions used for cell motility (Miki et al., (ii) fast migration typically on the order of tens of µm/min; and 1998; Steffen et al., 2004; Weiner et al., 2006; Veltman et al., (iii) the absence of specifc, high-affnity adhesions to the extra- 2012). In contrast, the involvement of WASP genes (particu- cellular environment. This independence from specifc molecular larly the two mammalian homologues WASP and N-WASP) in adhesions separates α-motility from the adhesion-based motility cell crawling is less clear. N-WASP is ubiquitously expressed of fbroblasts and epithelial cells. Furthermore, the use of pseu- in mammals and is dispensable for lamellipodia or flopodia dopods discriminates it from the fast bleb-based motility adopted formation by adherent fbroblasts (Lommel et al., 2001; Snap- by fbroblasts in environments that preclude adhesion formation per et al., 2001; Sarmiento et al., 2008), which has led many (Liu et al., 2015; Ruprecht et al., 2015). Some organisms using researchers to discount a role for any WASP protein in protru- α-motility may also use additional methods of generating forward sions or motility (Small and Rottner, 2010; Veltman and Insall, movement, such as contractility, retrograde fow, and/or blebbing 2010). Mammalian WASP, however, is expressed only in blood (Yoshida and Soldati, 2006; Lämmermann et al., 2008; Bergert et cells, where it has been shown to be involved in migration and al., 2012), but in this study, we focus on a single phenotype read- pseudopod formation (Badolato et al., 1998; Burns et al., 2001; ily observable in diverse species, including nonmodel organisms. Jones et al., 2002, 2013; Shi et al., 2009; Ishihara et al., 2012). Organisms with cells capable of α-motility appear Further evidence for a role of WASP in cell migration comes throughout the eukaryotic tree, and we hypothesize that this from the handful of papers studying WASP in nonmammalian form of locomotion refects a single, discrete process that arose cells (Veltman et al., 2012; Zhu et al., 2016; and see Tables S1 early in eukaryotic evolution and has been conserved. If this and S2 for an annotated bibliography of the 29 papers on WASP hypothesis is correct, then elements of this ancient process— and N-WASP relating to cell migration summarized here). The specifc molecules and mechanisms—may be conserved and use of WASP by highly motile cells but not by adherent fbro- Downloaded from still associated with cell crawling in distantly related organisms blasts may therefore refect unique requirements for α-motility. that use α-motility. Such molecular remnants would help to un- To understand the regulation of the actin cytoskeleton ravel the evolutionary history of cell locomotion and might en- during pseudopod formation, we exploit the diversity of organ- able us to predict the existence of specifc modes of motility in isms that use α-motility. By comparing the genomes of many poorly characterized species. Identifying genes associated with eukaryotes, we fnd that organisms with genes encoding both a process such as α-motility is not trivial because the core ma- WASP and SCAR make pseudopods, and organisms that do not chinery driving pseudopod formation (e.g., actin and the Arp2/3 build pseudopods have lost either or both Arp2/3 activators. We

complex) is shared with other cellular processes, including validate this molecular signature using a negative test (deplet- on June 21, 2017 some types of endocytosis (Winter et al., 1997). The partici- ing the protein disrupts pseudopod formation in well-studied pation of these proteins in multiple essential processes likely cells) as well as a positive test (a new prediction of α-motility explains their ubiquity in the eukaryotic family tree. Actin, for in a little-studied organism). Differentiating α-motility from example, is found in the genomes of all eukaryotes and, based slow/adhesive cell migration helps clarify the confusion over on phylogenetic analysis, is widely accepted to have been pres- WASP’s importance in cell motility and shifts the major ques- ent in the eukaryotic ancestor (Goodson and Hawse, 2002). The tion from whether WASP or SCAR is required for motility in a Arp2/3 complex is also highly conserved (Beltzner and Pollard, given single cell type to how WASP and SCAR work together to 2004). It is present in the genomes of all sequenced eukaryotes construct and maintain pseudopods in many species. The reten- except the parasitic protist Giardia intestinalis, whose lineage tion of WASP and SCAR by organisms that form pseudopods either split off before the evolution of the Arp2/3 complex or represents the frst molecular support, to our knowledge, for a lost it, depending on the placement of the root of the eukaryotic single origin of this widespread form of cell motility in an an- tree (Paredez et al., 2011). The ubiquity and multifunctionality cestor of extant eukaryotes (see Fig. 8 for summary). of actin and the Arp2/3 complex make them diffcult to use as markers for tracing the evolutionary history of α-motility. We therefore turned our attention to upstream regula- Results tors of actin assembly. There are several nucleation-promoting factors that stimulate branched actin network assembly by the Evolutionary retention of both WASP and Arp2/3 complex in response to various upstream cellular signals SCAR correlates with pseudopod formation (Rottner et al., 2010). Some of these Arp2/3 activators are re- To trace the evolutionary history of α-motility, we frst deter- stricted to specifc eukaryotic lineages, particularly multicellu- mined which sequenced eukaryotic organisms might use α-mo- lar animals that evolved JMY and WHAMM families of Arp2/3 tility. The obvious structural features associated with α-motility activators, whereas other activators are more widely distributed are dynamic, actin-flled pseudopods. In addition to α-motility, (Veltman and Insall, 2010; Kollmar et al., 2012). For example, some organisms use these structures for feeding. Pseudopods WASP and SCAR (also known as WAVE) are widely conserved used for feeding and α-motility share so many structural and Arp2/3 activators that respond to different signaling cascades signaling components that unless specifc receptors and/or prey (Rohatgi et al., 1999; Moreau et al., 2000; Koronakis et al., are known to be involved, they are largely indistinguishable 2011) and promote different levels of Arp2/3 activity (Zalev- (Heinrich and Lee, 2011). Therefore, we combed the literature sky et al., 2001). Multiple published phylogenetic analyses of for references to organisms with cells that form pseudopods for WASP and SCAR gene families suggest that both genes are an- feeding and/or α-motility. Eukaryotic phyla fall into at least six cient and likely to have been present in the eukaryotic ancestor large clades, and species with sequenced genomes and that form (Veltman and Insall, 2010; Kollmar et al., 2012). However, pseudopods can be found in most (see Fig. 8 and Table 1).

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On to this map of the phylogenetic distribution of pseu- and SCAR but forms Arp2/3-dependent phagocytic “food cups” dopods we overlaid the conservation of WASP and SCAR/ to engulf bacteria (Babuta et al., 2015). The absence of both genes WAVE genes, using a recently published manually curated da- indicates that Entamoeba must use another Arp2/3 activation sys- tabase of nucleation-promoting factors from genomes spanning tem for protrusions, an idea supported by its use of blebs and not eukaryotic diversity (Kollmar et al., 2012). Multiple analyses pseudopods for motility (Maugis et al., 2010). But the most glar- have concluded that both WASP and SCAR were present in the ing exception to the correlation was a pair of little-studied species last common ancestor of eukaryotes (Veltman and Insall, 2010; of chytrid fungi that retain both nucleation-promoting factors but Kollmar et al., 2012) and therefore argue that a lack of either are not known to build pseudopods: Allomyces macrogynus and gene refects loss during evolution. Batrachochytrium dendrobatidis (Bd). To understand whether these gene loss events reveal a We took a two-pronged approach to testing our hypothesis signifcant pattern, we compared the conservation of individ- that retention of WASP together with SCAR serves as a molec- ual nucleation-promoting factors across large evolutionary dis- ular signature of pseudopod formation. First, we took the more tances with the ability to assemble pseudopods. We identifed a traditional approach and confrmed that both genes are involved correlation between the conservation of WASP and SCAR and in pseudopod formation in mammalian cells. We followed this pseudopod formation (see Fig. 8 and Table 1). with an evolution-based approach by verifying the ability of this For example, no plant cells build pseudopods, and no se- molecular signature to predict the capacity for pseudopod for- quenced plant genomes contain a WASP orthologue. Similarly, mation in chytrid fungi. multicellular fungi—the dikarya—lack SCAR and are also not known to build pseudopods. Conversely, almost all sequenced ge- WASP and SCAR localize to the same nomes of Amoebozoan species (including dictyostelids) encode dynamic arcs within pseudopods of orthologues of WASP and SCAR, and almost all move with the human neutrophils help of dynamic, actin-rich pseudopods. A potential counterexam- Our evolutionary evidence indicates that WASP and SCAR may ple is the amoeba Entamoeba histolytica, which lacks both WASP both be required to build pseudopods. To test this hypothesis Downloaded from

Table 1. SCAR and WASP orthologues for indicated species (Kollmar et al., 2012) for protein sequences (except for orthologues in Sarracenia rosea, which we identified via BLA ST, NCBI identifiers indicated). See also Fig. 8.

Species Group SCAR WASP Pseudopod reference

H. sapiens Animals (opisthokont) WAVE1, WAVE2, WAVE3 WASP, N-WASP Ramsey, 1972

M. brevicollis Choanoflagellate MbWAVE (S. rosea: MbWASP (S. rosea: Pseudopod-based feeding has been observed in all on June 21, 2017 (orthologues in (opisthokont) XP_004994362) XP_004997564) choanoflagellate families (Pettitt et al., 2002); for S. rosea) detailed analysis of pseudopods of S. rosea see Dayel and King, 2014 C. owczarzaki Opisthokont CoWAVE CoWASP Hertel et al., 2002 A. nidulans Fungi (opisthokont) n.f. EnWASP n.f. (Representative dikaryon) B. dendrobatidis Fungi (opisthokont) Bad_bWAVE Bad_bWASP this work A. macrogynus Fungi (opisthokont) AlmWAVE AlmWASP_Aα, AlmWASP_Aβ, n.f. AlmWASP_Bα, AlmWAS PB_β D. discoideum Amoebozoan SCAR1 DdWASP Raper, 1935 E. histolytica Amoebozoan n.f. n.f. n.f. A. castellanii Amoebozoan AcWAVE AcWASP_A, AcWASP_B, Bowers and Korn, 1968 AcWASP_C T. trahens++ Apusozoa TctWAVE TctWASP Cavalier-Smith and Chao, 2010 A. thaliana Plant SCAR1, SCAR2, SCAR3, n.f. n.f. SCAR4, SCAR-like domain-containing protein P. ramorum Stramenopile (SAR) n.f. n.f. n.f. B. natans Rhizarian (SAR) n.f. BinWASP_A, BinWASP_B n.f. P. falciparum Alveolate (SAR) n.f. n.f. n.f. T. thermophila Alveolate (SAR) n.f. TtWASP_A, TtWASP_B n.f. E. huxleyi Haptophyte n.f. EmhWasp n.f. N. gruberi Heterolobosean NgWAVE_A, NgWAVE_B NgWASP_A, NgWASP_B, Fulton, 1970 NgWASP_C, NgWASP_D G. lamblia Diplomonad n.f. n.f. n.f. T. vaginalis Parabasalid Tv_aWAVE_A, Tv_aWAVE_B, Tv_aWasp_A, Tv_aWasp_B Kusdian et al., 2013 Tv_aWAVE_C, Tv_ aWAVE_D, Tv_aWAVE_E, Tv_aWAVE_F, Tv_ aWAVE_G, Tv_aWAVE_H, Tv_aWAVE_I, Tv_aWAVE_J, Tv_aWAVE_K

Example references for observations of pseudopods are given. Cases where genes or references to cells with active pseudopods were not found are indicated by “n.f.” See also Fig. 8.

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directly, we turned to human cell lines capable of forming the next section). Moreover, cells from WASP knockout mice pseudopods. HL-60 cells are derived from an acute myeloid also showed only a partial defect in gross cell motility in vivo leukemia (Collins et al., 1977) and retain many features of he- (Snapper et al., 2005). matopoietic cells, including expression of hematopoietic WASP In addition to the defect in pseudopod formation, ∼20% and the capacity to differentiate into fast-migrating neutrophils of WASP-KD cells formed large protrusions that tapered to with dynamic pseudopods (Collins et al., 1978). a point, reminiscent of a rhinoceros horn (Fig. 2, B, D, and To follow the dynamics of WASP localization in live cells, we F; Fig. S2; and Video 2; WASP-KD cells 11, 32, 33, 39, and created an HL-60 line stably expressing full-length WASP fused 42, for example). To verify its specifcity, we rescued this at the N terminus to the red fuorescent protein TagRFP-T. By “rhino” phenotype by expressing a functional WASP con- confocal fuorescence microscopy, TagRFP-WASP concentrated taining three silent mutations in the sequence targeted by the in two distinct locations within migrating HL-60 cells: punctate shRNA (Fig. 2 E). Additionally, a second shRNA that targets foci distributed throughout the cell and a broad zone near the a separate region of the WASP gene resulted in a signifcantly leading edge (Fig. S1 A). smaller effect on both WASP expression and the number of A previous study has shown that the SCAR regulatory cells with the rhino phenotype (not depicted). Immunofuores- complex localizes to fast-moving anterograde “waves” that cence combined with phalloidin staining of polymerized actin break against the leading edge of actively migrating HL-60 cells revealed that the aberrant rhino protrusions contained actin fl- (Weiner et al., 2007). This localization pattern is most easily ob- aments but lacked (Fig. 2 D). The expression of served using total internal refection fuorescence (TIRF) micros- a probe specifc for polymerized actin (mCherry fused to the copy, which illuminates a ∼100-nm thick region of the cell near calponin homology domain of ; Utr261; Burkel et al., the ventral surface (Axelrod, 1981). Using TIRF microscopy on 2007) revealed a highly dynamic and surprisingly hollow actin rapidly migrating HL-60 cells, we observed that TagRFP-WASP flament network inside the protrusions (Figs. 2 F and S2). concentrates near the leading edge in linear arcs that move in an This distribution, enriched near the membrane but depleted anterograde direction similar to previously observed patterns of from the core of the protrusion, is more reminiscent of cortical the SCAR regulatory complex (Weiner et al., 2007). actin networks than of flopodia, which are packed tightly with Downloaded from To see whether WASP and SCAR travel together in the actin bundles (Tilney et al., 1973). same waves, we introduced TagRFP-WASP into cells express- Because some WASP family proteins contribute to endo- ing YFP-Hem1, a core component of the SCAR regulatory cytosis (Naqvi et al., 1998; Merrifeld et al., 2004; Benesch et complex (Weiner et al., 2007). TIRF microscopy of these al., 2005), we investigated whether the defects in WASP-KD cells revealed that WASP and the SCAR regulatory complex cells are caused by reduction of endocytosis. In undifferenti- move together in the same dynamic, linear arcs (Fig. 1, A ated HL-60s, we observed no difference in transferrin receptor

and B; Fig. S1 B; and Video 1). Interestingly, however, the endocytosis and recycling between WASP-KD and control cells on June 21, 2017 localization patterns of the two are not identical, an observa- (Fig. S3 E). After differentiation into cells capable of making tion confrmed by quantifying WASP and SCAR localization pseudopods, WASP-KD HL-60s actually showed increased across the leading edge (Fig. S1 C). Spinning-disk confo- surface receptor densities (Fig. S3 D), receptor internalization cal microscopy indicated that WASP and SCAR colocalize (Fig. S3 B), and receptor recycling (Fig. S3 C) compared with throughout the growing pseudopods, not only at the ventral control cells. Therefore, we cannot attribute the WASP-KD phe- surface (Fig. 1 C). Within the resolution limits of our imag- notypes simply to a curtailment of endocytosis activity. ing, the localization patterns moved together, with neither protein consistently leading the other (Fig. S1 B and Video 1). WASP-depleted neutrophils polymerize less This dynamic localization pattern suggests that both WASP actin in response to chemoattractant and SCAR activate the Arp2/3 complex in leading-edge pseu- Addition of chemoattractant to nonpolarized (quiescent) HL-60 dopods, promoting assembly of the branched actin networks cells induced a burst of actin polymerization that drove polar- required for membrane protrusion. ization and pseudopod formation, nearly doubling the cell’s polymerized actin content within 30 s of stimulation. This re- WASP participates in pseudopod assembly sponse is already known to depend on the activity of the SCAR in neutrophils regulatory complex (Weiner et al., 2006). To determine what To investigate whether WASP is involved in pseudopod assem- role WASP might play in this explosive actin assembly, we bly by HL-60 cells, we generated anti-WASP shRNAs, expres- synchronized pseudopod formation by stimulating populations sion of which resulted in a >90% reduction of WASP protein of quiescent HL-60s with fMLP and then fxed and stained the but no obvious change in WAVE2 (Fig. 2 A). We next examined cells with phalloidin at different time points and analyzed total whether WASP-depleted (WASP-knockdown; KD; WASP-KD) polymerized actin content in each cell by confocal microscopy cells can form pseudopods. In a gradient of chemoattractant (the and FACS (Fig. 3, A and B). In the absence of chemoattractant, peptide fMet-Leu-Phe [fMLP]), wild-type HL-60 cells became the amount of polymerized actin in quiescent WASP-KD cells strongly polarized, with broad, actin-rich pseudopods used to was roughly equal to that in control cells. However, as reported rapidly move toward the source of chemoattractant (Fig. 2 B for SCAR-depleted cells (Weiner et al., 2006), WASP-KD cells and Video 2). Compared with control, 50% fewer WASP-KD had greatly reduced actin polymerization at both short (30 s) cells formed pseudopods (Fig. 2, B and C). Despite numerous and long times (3 min) after stimulation. This reduced actin attempts, we never succeeded in developing WASP-KD cell polymerization indicates that WASP, like SCAR, is central to lines in which this phenotype was 100% penetrant. Although it the explosive actin polymerization required for cell polarization is possible that this was caused by residual WASP protein, this and subsequent pseudopod formation. seems an insuffcient explanation because the WASP-KD cells To understand more about the pseudopods formed by some still capable of forming pseudopods were also aberrant (see WASP-KD cells, we analyzed confocal images of hundreds of

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Figure 1. WASP colocalizes with the SCAR complex at the leading edge of neutrophils. Microscopy of HL-60 cells expressing TagRFP- WASP and Hem-1–YFP, a component of the SCAR regulatory complex. (A) TIRF images of live HL-60 cells. Top: WASP localization in three sequential time points, overlaid on the far right (0 s in red, 20 s in green, and 40 s in blue). Middle: same sequence of images but for Hem-1. Bottom: overlay of WASP and Hem-1 at each time point. The plot shows line scans of normalized fluorescence intensity of WASP (red) and Hem-1 (green). The location for generating the line scans is shown in the adjacent image (white line). (B) Two additional examples of live HL-60 cells in TIRF and corre- sponding line scans (indicated by white lines in the images). (C) Spinning-disk confocal im- ages of two fixed HL-60 cells showing an axial slice through the middle of thick pseudopods. The slices shown were taken 3 µm above the coverslip. See also Fig. S1 (for additional im- ages and kymographs) and Video 1. For all cells, the direction of migration is to the left. Downloaded from on June 21, 2017

individual phalloidin-stained cells. Quantifcation of the actin 1.7 ± 0.4 µm/min (Fig. 4, A and B; and Video 2). This motility content in the subset of WASP-KD cells that make pseudopods defect was not limited to the rhino cells: when these cells were compared with control cells revealed that even WASP-KD cells excluded from the analysis, we still observed a signifcantly re- that appeared to make “normal” pseudopods contained about duced speed (6.7 ± 0.8 µm/min) compared with control cells half the quantity of polymerized actin (Fig. 3, C and D). (Fig. 4 B). We did not observe an effect of WASP depletion on directional persistence (Fig. 4 C). WASP depletion impairs neutrophil motility Because the chamber we used to measure directional mi- To determine the effect of WASP depletion on cell locomotion, gration was not precoated with fbronectin (or any other specifc we imaged HL-60 cells migrating through a chemoattractant molecule), we doubt this migration defect was caused by an gradient in a 5-µm-tall glass chamber (Millius and Weiner, integrin-mediated adhesion defect. We confrmed this by di- 2010). Tracking individual cells revealed a severe migration rectly testing adhesion to fbronectin-coated surfaces and found defect in WASP-KD cells (reported means ± SD of three bio- no signifcant difference between WASP-KD and control cells logical replicates): although control cells moved at 12 ± 0.8 µm/ (Fig. S3 A). We conclude that HL-60 cells use WASP along min, WASP-KD cells averaged 5.5 ± 1.5 µm/min, and cells with with SCAR (Weiner et al., 2006) for normal pseudopod forma- rhino protrusions were almost completely immotile, moving at tion and effcient α-motility.

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Figure 2. WASP is crucial for pseudopod formation of neutrophils. (A) Western blots showing WASP and WAVE2 expression in control HL-60 cells, cells expressing shRNA to WASP, and exogenous WASP with three silent mutations in the region corresponding to the shRNA (KD + rescue) or cells expressing anti-WASP shRNA and empty vector rescue control (KD + control). Approximately equal amounts of total protein were loaded in each lane, which was confirmed by using actin as a loading control. (B) Brightfield images of con- trol and WASP-KD cells. (C) KD of WASP using shRNA reduces the percentage of cells that have pseudopods. (D) Immunofluorescence of control and WASP-KD HL-60 cells showing microtubules (green, antibody stained), actin filaments (red, phalloidin stained), and DNA (blue, DAPI). Note the signature rhino pheno- type in the WASP-KD cell. See also Fig. S2. (E) Rescue of rhino protrusion phenotype by expression of shRNA-insensitive WASP as de- scribed in A. Note: no wild-type (WT) cells were observed to exhibit the rhino phenotype. (C and E) Means and SD (bars) of three bio- logical replicates (dots) with >130 cells total; p-values were obtained with two-tailed paired t tests. (F) Maximum projections of spinning-disk confocal stacks of living HL-60 cells with fluo- Downloaded from rescent probes specific for polymerized actin (mCherry fused to the calponin homology domain of Utrophin; Utr261). Insets are cross sections through the rhino horns at positions indicated by yellow lines, confirming that they are hollow with a shell of actin. See also Fig. S2 for a time lapse of the right-hand cell show- ing the dynamics of the rhino horn protrusion. on June 21, 2017

WASP and SCAR genes predict pseudopod Like other species of chytrid fungi, the lifecycle of Bd formation by chytrid fungi has two stages: a large (10–40 µm) reproductive zoosporan- A potential exception to the tight correlation between ac- gium, which releases a host of small (3–5 µm), motile, and tin-rich pseudopods and the genomic retention of WASP and fagellated zoospore cells (Longcore et al., 1999; Berger et al., SCAR were two deeply branching and little-studied species 2005). These infectious zoospores can form cysts beneath the of fungi: the chytrids A. macrogynus and Bd. These chytrid skin of an amphibian host that develop into new zoosporangia species contain genes encoding both WASP and SCAR but to complete the life cycle (Berger et al., 2005). We searched have not been reported in the literature to migrate using pseu- for α-motility in Bd zoospores because, unlike the sessile cyst dopods. We were, however, able to fnd references to pseudo- and zoosporangium, these free-swimming fagellates lack a cell pod formation by unsequenced infectious species related to wall and have been reported to assume nonuniform shapes with A. macrogynus (Catenaria anguillulae), which may use these dense cytoplasmic extensions (Longcore et al., 1999). structures for motility across the surface of its target host To restrict the fast-swimming Bd zoospores to the imaging (Deacon and Saxena, 1997; Gleason and Lilje, 2009). How- plane, we adhered zoospores to concanavalin A–coated glass. In ever, because chytrid fungi are not a monophyletic group but initial experiments, we observed only a small fraction (<1%) rather comprise multiple deeply branching clades that are es- of zoospores forming pseudopodlike protrusions. The rarity of timated to have diverged ∼800 million years ago (James et al., pseudopod-forming cells made us suspect that α-motility might 2006; Stajich et al., 2009), one cannot assume that distantly only occur during a short phase of the life cycle. We therefore related species share this capacity. Therefore, we used Bd as a enriched for cells of the same age by washing zoosporangia to predictive test of our hypothesis that WASP and SCAR genes remove previously released zoospores and collecting fagellates represent a marker for α-motility. released during the subsequent 2 h.

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Figure 3. WASP is crucial for explosive actin polym- erization during pseudopod formation in neutrophils. (A) Spinning-disk confocal images of polymerized actin of control and WASP-KD HL-60 cells after stimu- lation for the indicated time with chemoattractant (20 nM fMLP) stained with fluorescent phalloidin. (B) FACS quantification of actin polymerization in control (gray circles) and WASP-KD HL-60 cells (green triangles) after stimulation for the indicated time with chemoat- tractant stained with fluorescent phalloidin. 10,000 cells were counted for each sample, and means were normalized to time 0 for control cells within each ex- periment to control for detector variability. AU, arbi- trary unit. (C) Example spinning-disk confocal images of pseudopod-forming WASP-KD and control cells with polymerized actin stained with phalloidin (red) and DNA stained with DAPI (blue). (D) Quantification of phalloidin staining shown in C. Only cells with pseudopods were analyzed. Mean pixel values for z projections of image stacks was measured for each cell, and then the mean background pixel value was subtracted. Means and SD (bars) from three biological replicates (dots) are shown, with >150 cells total; the p-value was obtained from a one-tailed paired t test. Downloaded from on June 21, 2017

During the frst 6 h after release from the zoosporangium, a small molecule that sequesters actin monomers and inhibits ∼40% of zoospores created dynamic pseudopodlike protrusions the growth of actin polymers. Within minutes of adding 10 nM (Fig. 5, A and B; Fig. S4 A; and Video 3) that extended from the latrunculin B, nearly all pseudopods ceased growing and/or dis- cell body at a rate of 25 ± 9 µm/min (Fig. 5 C), consistent with appeared (Fig. 6, B and C). This effect was reversed within 1 h speeds expected for pseudopods (Chodniewicz and Zhelev, 2003; after removing the drug. Zhelev et al., 2004). Unlike blebs, these cellular protrusions To determine whether assembly of the pseudopodial actin were not spherical but were irregularly shaped and amorphous, network required the nucleation and branching activity of the similar to the actin-rich pseudopods of amoebae and neutrophils. Arp2/3 complex, we incubated zoospores with CK-666, a small To ensure that these crawling cells were not contaminat- molecule that inhibits actin nucleation by mammalian and fun- ing organisms, we obtained Bd cultures from three different gal Arp2/3 complexes (Nolen et al., 2009). Addition of 10 µM laboratories and observed similar pseudopods in each. CK-666 reduced the number of cells with active protrusions by To better investigate the morphology of these tiny pseudo- nearly 100%, an effect that was reversed by washing out the pods, we performed scanning electron microscopy on fxed cells drug (Fig. 6, D and E). These experiments reveal that protrusion (Figs. 5 D and S4 B) and observed a similar proportion of fag- of Bd pseudopods requires Arp2/3-dependent actin assembly. ellated zoospores with one or more thick protrusions. Each pro- trusion was ∼1 µm long and 1 µm wide, and many appeared to Chytrid zoospores use pseudopods for be composed of multiple discrete terraces (Figs. 5 D and S4 B). α-motility Although pseudopod-forming Bd cells adhered tightly to glass Chytrid pseudopods contain actin and surfaces coated with concanavalin A, they were not able to move require Arp2/3 activity or swim away from the site of initial attachment, and other coat- Using our assay to image chytrid zoospores, we next investigated ings did not promote any form of attachment (including collagen, whether extension of Bd pseudopods is driven by assembly of fbronectin, and human ; not depicted). Several types of branched actin networks as in other cells that crawl using α-mo- animal cells are known to migrate without specifc molecular ad- tility. We frst fxed the cells to preserve the actin cytoskeleton hesions in confned environments (Lämmermann et al., 2008; Liu and then stained them with fuorescent phalloidin, revealing a et al., 2015; Ruprecht et al., 2015). To test whether Bd zoospores thin shell of cortical actin surrounding the cell body and a dense might also be capable of migration in confned environments, we network of flamentous actin flling the pseudopod (Fig. 6 A). sandwiched cells between two uncoated glass coverslips held To test whether actin-flled chytrid pseudopods required apart by 1-µm-diameter glass microspheres and observed rapidly actin polymerization, we treated zoospores with latrunculin, migrating cells (Fig. 7 and Video 4). Obviously migrating cells

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Figure 4. WASP is crucial for neutrophil motility. (A) Worm plots showing the tracks of cells migrating up an fMLP chemoattractant gradient. Control cells are on the left, and WASP-KD cells are on the right, with cells exhibiting the rhino phenotype in red. Cells were imaged for 20 min and migration paths were over- laid, with time 0 at (0,0). The endpoint of each cell’s path is shown with a dot. See also Video 2. (B) De- pletion of WASP protein leads to reduced cell speed. The mean instantaneous speed for each cell in A is plotted as a dot color-coded by biological replicate to highlight the consistency from experiment to exper- iment. (C) Reduction of WASP protein leads to no sig- nificant change in directional persistence (the ratio of the Euclidean distance to the accumulated distance) of cells tracked in A. Means of the three replicates are displayed as horizontal lines; p-values were obtained from two-tailed paired t tests. Downloaded from on June 21, 2017

had a mean instantaneous speed of 19 ± 9 µm/min, with indi- muscle formation: like pseudopods, myoblast protrusions are an vidual cells averaging speeds >30 µm/min (Fig. 7 C), consistent actin-flled force generating machines that require both WASP with the measured rates of pseudopod extension (Fig. 5 C). The and SCAR (Sens et al., 2010). trajectories of these cells appeared fairly straight (Fig. 7 B), with Organisms without the capacity to crawl using pseudopods a mean directional persistence of 0.61 ± 0.25 (Fig. 7 D). turn out to have lost one or both of these nucleation-promoting Some pseudopod-forming zoospores retained fagella, factors (Fig. 8 and Table 1). The presence of genes encoding whereas other cells had clearly lost or resorbed their fagella and both WASP and SCAR, therefore, provides a molecular cor- strongly resembled free-living amoebae (Fig. 7 A and Videos 3 relate for a suite of behaviors that we call “α-motility.” The and 4). We also observed cells switching from crawling to fa- conservation and phylogeny of WASP and SCAR indicate that gellar motility and vice versa as well as cells rapidly retracting both were present in a common ancestor of living eukaryotes their fagellar axonemes into the cell body (Video 5). (Veltman and Insall, 2010; Kollmar et al., 2012). The power of the coconservation of WASP and SCAR as a genomic marker with the ability to identify cryptic pseudopod-forming organ- Discussion isms—together with cell biology evidence that both WASP and SCAR are required for α-motility in well-studied organisms— Our results reveal that across eukaryotic phyla, cells that con- argues that this widespread behavior arose from a single, an- struct actin-rich pseudopods and undergo fast, low-adhesion cient origin. It is formally possible that α-motility did not have a crawling have retained the Arp2/3 complex as well as two dis- single evolutionary origin, but that scenario would require both tinct activators of their actin nucleation activity: WASP and WASP and SCAR to be coopted together for pseudopod assem- SCAR/WAVE. This fnding is well supported by a recent paper bly multiple times during eukaryotic evolution. Because WASP implicating both WASP and SCAR in and SCAR are only two of a large number of Arp2/3 activators neuroblast cell migration (Zhu et al., 2016). In that system, (Rottner et al., 2010), we have no reason to believe that motility the phenotype of SCAR mutants is enhanced by the loss of would repeatedly converge on these two in particular. WASP, and both WASP and SCAR are found at the leading Metazoans and fungi, together with a handful of protists, edge of migrating neuroblasts in vivo. Our hypothesis is also form a major clade known as the “opisthokonts” (Fig. 8). Our consistent with a study of myoblast cell fusion events during identifcation of α-motility in a fungal species argues that the

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Figure 5. Genomic retention of both WASP and SCAR correctly predicts pseudopod for- mation by the infectious chytrid fungus Bd. (A) Time lapse showing examples of dynamic pseudopods from chytrid cells with (top) and without a flagellum (middle) or one cell of each (bottom). See also Video 3. (B) Percentage of cells with pseudopods within the first 6 h after release from zoosporangia. The mean and SD (bars) of four biological replicates (dots) is shown, with 3,782 cells total. (C) Pseudopod extension rates. The means and SD (bars) of the individual values (dots) combined from three biological replicates is shown. (D) Scan- ning electron micrographs of fixed chytrid zoospores. Brackets denote pseudopods and arrowheads denote flagella. See also Fig. S4 B for more examples. Downloaded from on June 21, 2017

ancestor of all the opisthokonts was capable of fast pseudo- (Video 5), a process that has been observed to take minutes in pod-associated crawling and that multicellular fungi represent other chytrid species (Koch, 1968). lineages that have lost α-motility (Fritz-Laylin et al., 2010). The α-motility of Bd flls an important gap in our under- Because cells of multicellular fungi are always encased in standing of the life cycle of this pathogen. As proposed for other rigid cell walls that would block pseudopods, selection pres- chytrid species (Gleason and Lilje, 2009), Bd zoospores may use sure to preserve gene networks specifc to pseudopod forma- pseudopods during the initial stages of their interaction with a tion was relieved, and the genes unique to this behavior were host either to move across epithelia or to crawl between epithe- subsequently lost. The fungi would therefore represent a large lial cells and invade the underlying stroma. Alternatively, our ob- eukaryotic lineage from which crawling motility has almost servation that newly hatched zoospores make more pseudopods completely disappeared. suggests that Bd may rely on α-motility to crawl along or within Images from an earlier study revealed individual Bd the epithelial surface to uninfected tissues or to exit the host. zoospores with irregular shapes and cytoplasmic extensions Imaging chytrid zoospores provided key evidence for the (Longcore et al., 1999). Actin-driven pseudopod formation involvement of WASP and SCAR in a conserved mode of cell and cell motility, however, were not previously described in migration, but further exploration of WASP and SCAR function Bd cells, in part because this species was discovered quite re- in Bd is hampered by several factors. First, the zoospores are cently (Longcore et al., 1999), and relatively few studies have small enough to pose challenges to live-cell imaging. Second, been devoted to its cell biology. In addition, Bd zoospores are the absence of genetic tools makes it impossible to fuorescently quite small (<5 µm) and highly motile, so visualizing their label or deplete proteins in live zoospores. Finally, the lack of tiny (∼1 µm) pseudopods requires physical confnement and potent and specifc inhibitors of WASP and SCAR (Guerriero high-resolution microscopy. Finally, because only recently and Weisz, 2007; Bompard et al., 2008) precludes chemical dis- released zoospores crawl, synchronization of cell cultures ruption of their activity. was crucial. These advances not only enabled us to observe Several protein families are known to activate the Arp2/3 α-motility but also revealed Bd zoospores retracting their fa- complex, including WASP, SCAR, WASH, JMY, and WHAMM gella by coiling the entire axoneme into the cell body in <1 s (Rottner et al., 2010). A conventional explanation for the multi-

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Figure 6. Chytrid pseudopods are actin filled and re- quire both actin polymerization and Arp2/3 activity. (A) Fixed chytrid cells with and without a flagellum (arrow). Staining with fluorescent phalloidin reveals a thin shell of cortical actin surrounding the cell body and a dense network of polymerized actin filling the pseudopods (brackets). DIC, differential interference contrast. (B) Two examples of chytrid cells with pseu- dopods that lose them when treated with 10 nm latrun- culin B, an inhibitor of actin polymerization. Dynamic pseudopods (brackets) return after drug washout. (C) Quantification of reversible inhibition of pseudopods by latrunculin B (Lat B). Only cells that were mak- ing pseudopods before treatment and that were not washed away during the experiment were counted. (D) Two examples of chytrid cells with pseudopods that lose them when treated with 10 µm CK-666, an inhibitor of Arp2/3 activity. Pseudopods return after drug washout. (E) Quantification of reversible inhibition of pseudopods by CK-666. Only cells that were making pseudopods before treatment and that were not washed away during the experiment were counted. Symbols are means from three biological replicates, each with at least 29 (C) or 17 (E) cells. P-values were obtained from two-tailed paired t tests. Downloaded from on June 21, 2017

plicity of Arp2/3 activators is that each promotes the construc- feedback that drives the explosive actin polymerization required tion of an actin network with a unique cellular function and/or for α-motility, but which can also result in spurious pseudopod location. However, the evolutionary connection between WASP, formation (Chung et al., 2000). We propose that the use of two SCAR, and pseudopod formation suggests that nucleation-pro- distinct activation systems, WASP and SCAR, reduces the prob- moting factors can work together, in this case to drive the ex- ability of errant pseudopod formation by using both activators plosive actin polymerization required for α-motility. Indeed, we to raise Arp2/3 activity above the threshold required for ro- fnd that WASP and SCAR colocalize at the leading edge of bust pseudopod formation. crawling neutrophils, and WASP depletion results in aberrant Under this coincidence detection model, an occasional pseudopods and reduced motility similar to reported effects of spike in the local activity of either WASP or SCAR may be suf- SCAR depletion (Weiner et al., 2006). But why have multiple fcient to trigger pseudopod formation, but activation of both distinct Arp2/3 activators instead of simply increasing the con- should be more effcient at driving explosive actin assembly. centration of one of them? The answer may lie in the positive This fts our observation that WASP depletion results in a subset

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Figure 7. Genomic retention of both WASP and SCAR correctly predicts α-motility in the in- fectious chytrid fungus Bd. (A) Example chytrid zoospores with and without a flagellum migrat- ing when confined between 1-µm spaced glass coverslips. See also Video 4. (B) Worm plots showing the tracks of 26 migrating chytrid zoospores with migration paths overlaid and with time 0 at (0,0). The endpoint of each cell’s path is shown with a dot. Only those cells ob- viously migrating were tracked, and cells were tracked for the duration of their movement. (C) Mean instantaneous speed of cells tracked in B. (D) Directional persistence (the ratio of the Euclidean distance to the accumulated distance) of cells tracked in B. Means and SD (bars) of the individual values (dots) combined from three biological replicates are shown. Downloaded from on June 21, 2017

of cells capable of pseudopod formation but with less polym- N-WASP for flopodia or sheet-like surface-adhered lamelli- erized actin (Fig. 3, C and D) and reduced migration speeds podia (Lommel et al., 2001; Snapper et al., 2001; Sarmiento (Fig. 4 B) and explains previous studies showing the incomplete et al., 2008). Such papers have been cited as proof that all penetrance of WASP and SCAR phenotypes (Blagg et al., 2003; WASP family proteins are dispensable for protrusions in gen- Myers et al., 2005; Snapper et al., 2005; Weiner et al., 2006; eral (Small and Rottner, 2010). Such generalizations depend Veltman et al., 2012). This model may also explain the rhino on two assumptions: that N-WASP and WASP have the same phenotype: without the additional Arp2/3 activation provided molecular function and that the adherent motility of fbroblasts by WASP, the resulting sparse actin networks may collapse and and α-motility use the same molecular pathways. However, a coalesce into the observed horn-shaped structures. This idea recent molecular replacement study showed that WASP and the is supported by studies of the upstream activators of WASP ubiquitously expressed N-WASP have different functions and (Cdc42) and SCAR (Rac) that indicate that Rac mediates a pos- cannot compensate for each other (Jain and Thanabalu, 2015). itive feedback loop required for leading-edge formation but that Furthermore, when one considers the large body of mamma- the stability of the resulting protrusion requires Cdc42 (Srini- lian WASP literature in the light of distinct modes of motility, a vasan et al., 2003; Stradal and Scita, 2006). simple pattern emerges: cell types that do not natively express This model also clears up confusion in the feld regard- WASP do not make pseudopods (although they may make sur- ing WASP’s role in cell migration. Our results are supported face-bound lamellipodia, linear flopodia, or adhesive structures by papers showing that blood cells rely on WASP for effcient called podosomes); WASP is only expressed in blood cells, and cell migration (Binks et al., 1998; Zicha et al., 1998; Anderson these cells use WASP for pseudopod-based migration (see Ta- et al., 2003; Snapper et al., 2005; Zhang et al., 2006; Blundell bles S1 and S2 for an annotated summary of WASP/N-WASP et al., 2008; Dovas et al., 2009; Kumar et al., 2012; Worth et literature). The predominant view that WASP is not involved al., 2013) and others suggesting that WASP plays a direct role in cell migration demonstrates the peril of assuming that in- in protrusion formation, including pseudopods (Badolato et sights based on adhesion-dependent cell motility apply to other al., 1998; Burns et al., 2001; Jones et al., 2002, 2013; Shi et modes of cell crawling. al., 2009; Ishihara et al., 2012). However, these data have been In addition to motility, Arp2/3 activators have been shown overshadowed by studies showing that fbroblasts do not require to play roles in other cellular processes, including endocytosis

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and endocytosis, both SCAR and WASP protein families have been shown to interact with endocytosis pathways (Badour et al., 2007; Gautier et al., 2011). Accordingly, we found that WASP-defcient HL-60 cells maintained normal receptor inter- nalization and recycling (Fig. S3 E) until pseudopod activity was activated by differentiation into neutrophils. After differen- tiation, in addition to being defective in building pseudopods, WASP-KD cells exhibited increased endocytosis and recep- tor recycling (Fig. S3, B–D). This is consistent with the idea that actin-mediated endocytosis is more effcient when cells are not making pseudopods. Although a large number of eukaryotes make pseudopods (Fig. 8 and Table 1), only two lineages are currently genetically tractable: animals and dictyostelids. Studies in both confrm that pseudopod formation involves both WASP (Badolato et al., 1998; Burns et al., 2001; Jones et al., 2002, 2013; Shi et al., 2009; Ishihara et al., 2012) and SCAR (Miki et al., 1998; Stef- fen et al., 2004; Weiner et al., 2006; Veltman et al., 2012), in contrast to some animal cell types that may only require SCAR for lamellipodia-based migration (Snapper et al., 2001; Bryce et al., 2005; Misra et al., 2007; Sarmiento et al., 2008). With our discovery of α-motility in the fungus Bd, we conclude that both proteins have been conserved together to facilitate this evolu- tionarily ancient mode of cell motility. Downloaded from

Materials and methods

Antibodies and Western blotting Rabbit anti-WASP antibody (sc-8353) was from Santa Cruz Biotech-

Figure 8. Only organisms that make pseudopods retain both WASP and nology, Inc., as was the goat anti-WAVE2 (sc-10394). Mouse anti- on June 21, 2017 SCAR genes. Diagram showing the relationships of extant eukaryotes (DM1A) was from Sigma-Aldrich, and mouse anti-actin (based on a study by Fritz-Laylin et al., 2010) with the presence or ab- sence of SCAR (blue) and WASP (green) genes from complete genome (JLA20) was from EMD Millipore. Western blotting was conducted sequences as described previously (Kollmar et al., 2012). Each represen- using standard protocols and HRP-conjugated secondary antibodies tative organism whose genome was used for the analysis is listed to the (Jackson ImmunoResearch Laboratories, Inc.). right. For groups with similar morphological and sequence patterns, a single species is used. For example, there is no known plant species that forms pseudopods or retains the WASP gene, so only a single species is Generation of HL-60 cell lines shown (Arabidopsis thaliana); similarly, Aspergillus nidulans represents all HL-60 lines were derived from CCL-240 (ATCC) and were grown in dikarya. See Kollmar et al. (2012) for additional sequence information. RPMI 1640 medium supplemented with 15% FBS, 25 mM Hepes, and An amoeba glyph indicates organisms that build pseudopods. Outlined 2.0 g/liter NaHCO3 and were grown at 37°C and 5% CO2. WASP-KD rectangles indicate a lack of an identifiable gene. See Table 1 for citations was achieved using Sigma-Aldrich’s Mission Control shRNA vector and full species names. *, Although we were not able to find a reference to pseudopod formation in A. macrogynus, a relative (C. anguillulae) does (TRCN0000029819; hairpin sequence 5′-CCG GCG AGA CCT CTA AAC assemble pseudopods used for motility (Deacon and Saxena, 1997; Glea- TTA TCT ACT CGA GTA GAT AAG TTT AGA GGT CTC GTT TTT-3′) son and Lilje, 2009). Because of this and the conservation of both WASP with corresponding control vector expressing anti-GFP shRNA and SCAR in Bd (highlighted in bold), we correctly predicted this species is (SHC005; hairpin sequence 5 -CCG GCG TGA TCT TCA CCG ACA also capable of pseudopod formation. ++, These species form pseudopods ′ for feeding rather than motility. The question mark indicates uncertainty AGA TCT CGA GAT CTT GTC GGT GAA GAT CTT TTT-3′). Lentivirus regarding the structure of the protrusions for phagocytosis in E. histolytica was produced in HEK293T grown in six-well plates and transfected (see the Evolutionary retention of both WASP and SCAR correlates with with equal amounts of the lentiviral backbone vector (either protein ex- pseudopod formation section). The time of divergence of extant eukary- pression vector derived from pHRSIN-CSGW [Demaison et al., 2002] otic groups has been estimated to be 1.1–2.3 billion years ago (bya; or shRNA expression vectors described in the previous sentence), Chernikova et al., 2011; Parfrey et al., 2011; Knoll, 2014) and has been predicted to have possessed both WASP and SCAR gene families (Kollmar pCMVΔ8.91 (encoding essential packaging genes), and pMD2.G (en- et al., 2012) and therefore may have built pseudopods. coding VSV-G gene to pseudotype virus). pHRS IN-CSGW and pack- aging vectors were obtained from R. Vale (University of California, (Naqvi et al., 1998; Merrifeld et al., 2004; Benesch et al., San Francisco, San Francisco, CA). After 48 h, the supernatant from 2005). The relationship between cell motility and endocyto- each well was removed, centrifuged at 14,000 g for 5 min to remove sis is complex and not completely understood (Traynor and debris, and then incubated with ∼106 HL-60 cells suspended in 1 ml Kay, 2007; Schiefermeier et al., 2011). Rapid pseudopod ex- complete RPMI for 5–12 h. Fresh medium was then added, and the tension requires not only a large quantity of actin polymeriza- cells were recovered for 3 d to allow for target protein or shRNA ex- tion (Weiner et al., 2006) but also increases membrane tension pression. TagRFPt-WASP fusion was cloned by frst swapping out (Diz-Muñoz et al., 2016), both of which counteract effcient eGFP for TagRFP-T (Shaner et al., 2008) in the pHRS IN-CSGW clathrin- and actin-mediated endocytosis (Boulant et al., 2011). by PCR amplifying TagRFP-T with 5′-CCC GGG ATC CAC CGG Despite this apparent dichotomy between protrusion formation TCG CCA CCA TGG TGT CTA AGG GCG AAG AGC TGA TTA AGG-3′

1684 +$#t70-6.&t/6.#&3t Published May 4, 2017

and 5′-GAG TCG CGG CCG CTT TAA CTA GTC CCG CTG CCC TTG al., 2010). Image analysis was performed with the ImageJ bundle Fiji TAC AGC TCG TCC ATG CCA TTA AGT TTG TGC CCC-3′ primers and (National Institutes of Health; Schindelin et al., 2012). All imaging was then cloning the resulting PCR product into pHRS IN-CSGW, using done at room temperature. NotI and BamHI to produce the pHR-TagRFP-T vector. Then, the WASP open reading frame was PCR amplifed from cDNA (NCBI Quantification of actin polymerization by flow cytometry accession number BC012738) using 5′-GCA CTA GTA TGA GTG GGG HL-60 cells were depolarized in serum-free medium supplemented GCC CAA TGG GAG GAA-3′ and 5′-AAG CGG CCG CTC AGT CAT with 2% low-endotoxin BSA (Sigma-Aldrich) for 1 h at 37°C and 5% CCC ATT CAT CAT CTT CAT CTT CA-3′ primers and then cloned into CO2 before simulation with 20 nM fMLP for the indicated time. Cells the pHR-TAG RFP-T backbone using NotI and SpeI to result in a sin- were immediately fxed with 4% paraformaldehyde in cytoskeleton gle open reading frame containing TagRFPt, a fexible linker (amino buffer on ice for 20 min, stained with PBS supplemented with 2% BSA, acids GSGTS) followed by full-length WASP. The WASP shRNA res- 0.1% Triton X-100, and 66 nM Alexa Fluor 488–conjugated phalloidin cue vector was cloned by inserting a P2A cleavage site (Kim et al., (A12379; Molecular Probes) for 20 min, and then washed thrice with 2011) between the linker and a WASP open reading frame edited with PBS supplemented with 0.1% Tween-20 before FACS analysis. site-directed mutagenesis to contain three silent mutations within the shRNA-targeting region (5′-CGA GAC CTC TAA ACT TAT CTA-3′ Cell adhesion assay was changed to 5′-CGAaACC TCT AAgCTcATC TA-3′; silent mu- Differentiated control and WASP-KD HL-60 cells were each stained tations in lowercase). A corresponding control vector was designed with either green or blue acetoxymethyl ester dyes (CellTrace Cal- to express TagRFPt with the fexible linker but no portion of WASP. cein green and blue; Thermo Fisher Scientifc), and equal numbers The Hem1-YFP line (R6) was previously described (Weiner et al., were mixed and allowed to attach to fbronectin-coated cover glass– 2007) and was developed by selecting HL-60 cells expressing both an bottomed 96-well plates for 30 min at 37°C. One set of wells was gen- shRNA-targeting native Hem1 and a YFP-tagged version of Hem-1 al- tly washed three times with fresh media. 100 random locations within lowing fuorescence imaging of the SCAR complex in HL-60 cells. the well were immediately imaged, and the percentage of remaining shRNA lines were selected by puromycin (1 µg/ml for at least 1 wk) cells was calculated and normalized to control unwashed wells. Downloaded from and fuorescent cell lines by FACS. HL-60 cells were differentiated by treatment with 1.3% DMSO for 5 d. Transferrin uptake endocytosis assays 5 × 106 differentiated HL-60 cells were washed twice with ice-cold Cytometry serum-free growth medium (SF), transferred to 37°C for 5 min (to FACS analysis was performed on a FACSCalib ur analyzer (BD), Data clear surface-bound transferrin), and chilled on ice for 1 min. An were analyzed with FlowJo software (Tree Star), and dead cells were equal volume of cold SF supplemented with 100 µg/ml Alexa Fluor gated out using forward and side scatter for all analyses. A FACS Aria 488–conjugated transferrin (T-13342; Molecular Probes; McGraw

II was used for sorting. All FACS analysis was performed at the Univer- and Subtil, 2001) was added and incubated on ice for 10 min. Cells on June 21, 2017 sity of California, San Francisco, Laboratory for Cell Analysis. were then washed twice with cold SF medium, transferred to 37°C for the indicated time period, washed twice with ice-cold acid buffer Imaging (8.76 g NaCl, 9.74 g 2-morpholin-4-ylethanesulfonic acid in 900 ml, EZ-TAX IScan (Effector Cell Institute) analysis of HL-60 cell migration pH to 4.0, and water to 1 liter), fxed in 4% paraformaldehyde in 1× between glass surfaces was conducted as previously described (Millius PBS for 20 min, and washed twice more with ice-cold PBS before and Weiner, 2010), and cell migration analyzed using the Chemotaxis immediate FACS analysis. and Migration Tool (Ibidi). Fixed HL-60 cells were imaged with a 100× 1.40 NA oil Plan Apo objective on a motorized inverted microscope Motility of chytrid zoospores (Ti-E; Nikon) equipped with a spinning disk (CSU22; Yokogawa Elec- The Bd strain JEL423 was obtained from J. Longcore (University of tric Corporation) and an electron-multiplying charge-coupled device Maine, Orono, ME) and grown in 1% tryptone broth or on agar plates camera (Evolve; Photometrics). Live TIRF images were acquired by (1% tryptone and 2% agar) at 25°C. Before imaging, cultures were plating HL-60 cells on cover glass cleaned by a 30 min incubation synchronized by either washing three times in 1% tryptone and har- in 3 M NaOH and then four washes with PBS, pH 7.2, coated for 30 vesting zoospores 2 h later (for liquid cultures) or by f ooding agar min with 100 µg/ml bovine fbronectin (F4759; Sigma-Aldrich) resus- plates with ∼2 ml water (for agar plates) passed through a 40-µm fl- pended in PBS. TIRF microscopy images were acquired on an inverted ter (Falcon), were collected by centrifuging at 1,200 g for 5 min, and microscope (TE2000; Nikon) equipped with a 60× or 100× 1.49 NA oil then were resuspended in Bonner’s Salts (Bonner, 1947). Cell motility Apo TIRF objective and an electron-multiplying charge-coupled de- was imaged by sandwiching cells between a number 1.5 glass coverslip vice (iXon+; Andor Technology) using previously described imaging and glass slide (cleaned by sonicating in pure water) separated using conditions (Weiner et al., 2007). In brief, differentiated HL-60 cells 1-µm glass microspheres (Bangs Laboratories). Coverslips and glass were plated on fbronectin-coated coverslips in modifed HBSS supple- slides were sonicated in deionized water and dried. Cells were treated mented with 0.2% serum albumin and imaged with 100-ms exposures with either 10-µM CK-666 (Sigma-Aldrich) or 10-nM latrunculin B every 1–2 s. Fixed chytrid cells were imaged using an inverted Ti-E (Sigma-Aldrich) while being adhered to concanavalin A–coated glass. microscope equipped with a spinning-disk confocal system with 33-µm For visualization of polymerized actin: 400 µl fxation buffer (50 mM pinholes and a 1.8× tube lens (Spectral Diskovery), a 60× 1.49 NA Apo cacodylate buffer, pH 7.2) supplemented with 4% glutaraldehyde was TIRF objective (Nikon), and a complementary metal oxide semicon- added to 100 µl cells attached to a concanavalin A–coated coverslip and ductor camera (Zyla 4.2; Andor Technology). Differential interference incubated for 20 min at 4°C. Samples were quenched with tetraborohy- contrast microscopy was performed on a Ti-E inverted microscope dride, permeabilized with 0.1% Triton X-100, incubated for 20 min with with a light-emitting diode illuminator (TLED; Sutter Instrument) Alexa Fluor 488–labeled phalloidin (Invitrogen), rinsed four times, and and a 100× 1.49 NA Apo TIRF objective. Images were acquired on imaged described in the Imaging section. Samples for scanning elec- a complementary metal oxide semiconductor camera. All microscopy tron microscopy were fxed as for visualization of polymerized actin, hardware was controlled with Micro-Manager software (Edelstein et stained with osmium tetroxide, dehydrated, critical point dried, and

&WPMVUJPOBSZSPMFTPG8"41BOE4$"3JONPUJMJUZt'SJU[-BZMJOFUBM 1685 Published May 4, 2017

Au/Pd sputter–coated according to standard protocols and then were species. J. Mol. Biol. 336:551–565. http ://dx .doi .org /10 .1016 /j .jmb .2003 imaged using a scanning electron microscope (S-5000; Hitachi) in the .12 .017 Benesch, S., S. Polo, F.P.L. Lai, K.I. Anderson, T.E.B. Stradal, J. Wehland, and University of California, Berkeley, Electron Microscopy Laboratory. K. Rottner. 2005. N-WASP defciency impairs EGF internalization and actin assembly at clathrin-coated pits. J. Cell Sci. 118:3103–3115. http:// Online supplemental material dx .doi .org /10 .1242 /jcs .02444 Fig. S1 shows how WASP localizes to pseudopods of migrating neu- Berger, L., A.D. Hyatt, R. Speare, and J.E. Longcore. 2005. Life cycle stages of the amphibian chytrid Batrachochytrium dendrobatidis. Dis. Aquat. trophils. Fig. S2 shows how WASP depletion in neutrophils leads to Organ. 68:51–63. http ://dx .doi .org /10 .3354 /dao068051 dynamic rhino protrusions. Fig. S3 shows how WASP is not required Bergert, M., S.D. Chandradoss, R.A. Desai, and E. Paluch. 2012. Cell mechanics for adhesion or endocytosis by HL-60 cells. Fig. S4 shows additional control rapid transitions between blebs and lamellipodia during migration. examples of chytrid pseudopods. Video 1 shows TIRF microscopy of Proc. Natl. Acad. Sci. USA. 109:14434–14439. http ://dx .doi .org /10 .1073 /pnas .1207968109 HL-60 cells. Video 2 shows chemotaxis of HL-60 cells. Video 3 shows Binks, M., G.E. Jones, P.M. Brickell, C. Kinnon, D.R. Katz, and A.J. Thrasher. time lapses of chytrid zoospores making pseudopods. Video 4 shows 1998. Intrinsic dendritic cell abnormalities in Wiskott-Aldrich syndrome. chytrid cells crawling. Video 5 shows an example of a chytrid zoospore Eur. J. Immunol. 28:3259–3267. http ://dx .doi .org /10 .1002 /(SICI)1521 retracting its fagellum. Tables S1 and S2 summarize the literature on the -4141(199810)28 :10<3259::AID-IMMU3259>3.0.CO;2-B roles of WASP and N-WASP in protrusion formation and cell motility. Blagg, S.L., M. Stewart, C. Sambles, and R.H. Insall. 2003. PIR121 regulates pseudopod dynamics and SCAR activity in Dictyostelium. Curr. Biol. 13:1480–1487. http ://dx .doi .org /10 .1016 /S0960 -9822(03)00580 -3 Acknowledgments Blundell, M.P., G. Bouma, Y. Calle, G.E. Jones, C. Kinnon, and A.J. Thrasher. 2008. Improvement of migratory defects in a murine model of Wiskott- Aldrich syndrome gene therapy. Mol. Ther. 16:836–844. http ://dx .doi .org We would like to thank Jasmine Pare for help with cell culture and /10 .1038 /mt .2008 .43 HL-60 cell migration assays and Joyce Longcore and Jason Stajich for Bompard, G., G. Rabeharivelo, and N. Morin. 2008. Inhibition of cytokinesis by helpful discussions. wiskostatin does not rely on N-WASP/Arp2/3 complex pathway. BMC This work was supported by the National Institute of General Cell Biol. 9:42. http ://dx .doi .org /10 .1186 /1471 -2121 -9 -42 Medical Sciences of the National Institutes of Health (R01-GM061010 Bonner, J.T. 1947. Evidence for the formation of cell aggregates by chemotaxis in the development of the slime mold Dictyostelium discoideum. J. Exp. Downloaded from to R.D. Mullins), by the Howard Hughes Medical Institute Investigator Zool. 106:1–26. http ://dx .doi .org /10 .1002 /jez .1401060102 program (R.D. Mullins), and by a postdoctoral fellowship from the Boulant, S., C. Kural, J.-C. Zeeh, F. Ubelmann, and T. Kirchhausen. 2011. Helen Hay Whitney Foundation supported by the Howard Hughes Actin dynamics counteract membrane tension during clathrin-mediated Medical Institute (L.K. Fritz-Laylin). endocytosis. Nat. Cell Biol. 13:1124–1131. http ://dx .doi .org /10 .1038 / ncb2307 The authors declare no competing financial interests. Bowers, B., and E.D. Korn. 1968. 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1688 +$#t70-6.&t/6.#&3t Supplemental material JCB

Fritz-Laylin et al., https ://doi .org /10 .1083 /jcb .201701074

Figure S1. WASP localizes to pseudopods of migrating neutrophils. (A) Maximum-projection image of localization of TagRFP-WASP and Hem-1–YFP (a member of the SCAR regulatory complex) expressed in differentiated HL-60 cells fixed while migrating on a fibronectin-coated surface. (B) Kymographs of pseudopods of two live HL-60 cells showing colocalization over time of TagRFP-WASP and Hem-1–YFP patterns. Time is from left (T = 0) to right, and direction is from bottom (inside cell) to top (outside cell). See Video 1 for the cells from which these kymographs were generated. (C) Line scans following the contour of the leading edge of each individual cell shown Fig. 1 (A and B) showing that TagRFP-WASP (red) partially colocalizes with Hem-1–YFP (green). THE JOURNAL OF CELL BIOLOGY CELL OF JOURNAL THE

Figure S2. WASP depletion in neutrophils leads to dynamic rhino protrusions. Time-lapse microscopy of a live WASP-KD HL-60 cell with aberrant rhino protrusions, with polymerized actin visualized using Utrophin261-mCherry. Time is shown in min:s, and an immobile arrowhead highlights protrusion dynamics. The 01:15 time point of this cell is shown in Fig. 2 F on the right.

Evolutionary roles of WASP and SCAR in motility t'SJU[-BZMJOFUBM 4 Figure S3. WASP is not required for adhesion or endocytosis by HL-60 cells. (A) WASP-KD does not significantly reduce HL-60 cells’ ability to adhere to fibronectin-coated surfaces. Bars represent means from three biological replicates normalized to an internal control in each experiment. The p-value was obtained from a two-tailed paired t test. At least 1,000 cells were counted for each internal control. (B) Steady-state endocytosis was measured for WASP-KD (green) and control cells (gray) by incubating cells for 10 min at 37°C with fluorescent transferrin and then immediately washing with ice-cold acid buffer to remove surface-bound transferrin. Cells were then fixed, and endocytosed transferrin was quantified by FACS analysis. (C) Quantification of actin-mediated endocytosis and receptor recycling in differentiated (neutrophil-like) control (black circles) and WASP-KD HL-60 cells (green squares). Cells were incubated with fluorescent transferrin, placed at 37°C for the indicated time, washed with ice-cold acid buffer to remove surface-bound transferrin, and fixed for FACS analysis. (D) Transferrin receptor density was measured by incubating cells at 37°C in serum-free medium (to remove surface-bound transferrin), chilling cells, and then incubating on ice with fluorescent transferrin. Cells were then washed with PBS to remove unbound transferrin and fixed. Surface-bound transferrin was then quantified by FACS analysis. (B and D) Values were normalized to the percent control within each of three independent experiments, with 10,000 cells analyzed for each sample. (E) Quantification of actin-mediated endocytosis and receptor recycling in undifferentiated con- trol (black squares) and WASP-KD HL-60 cells (green triangles). Cells were prepared as in C. Note that the recycling assays are normalized. For comparing absolute amounts of material endocytosed, refer to B. Within each experiment, samples for each cell line were normalized to time 0. Means and SD for three independent experiments are shown, with 10,000 cells analyzed for each sample. AU, arbitrary unit.

S18 +$# t Figure S4. Additional examples of chytrid pseudopods. (A) Differential interference contrast image of a representative field of synchronized Bd zoo- spores. Asterisks highlight cells with obvious pseudopods. Rapidly swimming flagellate cells are blurred. (B) Additional examples of scanning electron micrographs of chytrid zoospores.

Evolutionary roles of WASP and SCAR in motility t'SJU[-BZMJOFUBM S19 Video 1. Five examples of time-lapse videos showing TIRF microscopy of live differentiated HL-60 neutrophil cells expressing Hem-1–YFP and TagRFP-WASP. Hem-1–YFP is on the top and is green in the overlay; TagRFP-WASP is in the middle and is red in the overlay. White lines indicate positions for kymographs in Fig. S1 B. One frame was acquired every 2 s, and video is displayed at 10 frames per second.

Video 2. Migration of control differentiated HL-60 neutrophil cells and of differentiated WASP-KD HL-60 cells in a 2D environ- ment (EZ-TAX IScan assay). Cells expressing control shRNA migrating in a chemoattractant gradient (source is at top) between two glass surfaces with 5-µm spacing. All cells initially within the field of view were manually tracked as shown. Many WASP-KD cells exhibited the rhino phenotype, and their motility was effectively abolished (e.g., cells 11, 32, 33, 39, and 42). One frame was acquired every 20 s, and video is displayed at 15 frames per second.

Video 3. Time-lapse videos showing the representative examples of Bd chytrid zoospores with pseudopods imaged using dif- ferential interference contrast microscopy pictured in Fig. 4 B, including a cell with flagella (bottom left), a cell without a flagellum (bottom right), and one cell of each (top). One frame was acquired per second, and video is displayed at four frames per second.

Video 4. Examples of Bd chytrid cells crawling between two glass coverslips separated by 1-µm glass beads. See also Fig. 5. One frame was acquired per second, and video is displayed at 20 frames per second.

Video 5. Time-lapse imaging showing an example of flagellar retraction by Bd chytrid zoospores. One frame was acquired every 0.1 s, and video is displayed at 20 frames per second.

4 +$# t Table S1. Fast/blood cell (expressing WASP)

References (PMID) Cell type or types Finding

Jones et al., 2002 Macrophages (from blood Migrating macrophages that lack WASP fail to form actin-rich protrusions. WASP-deficient cells moved (11950596) of human Wiskott-Aldrich aberrantly and did not have directionality toward a chemoattractant. See Fig. 2. syndrome patients) Burns et al., 2001 Dendritic cells (from blood Expressing WASP restores actin- and WASP-filled protrusions. Arp2/3 is also shown to be enriched in the (11493463) of human Wiskott-Aldrich protrusions. “Persistent broad, leading-edge lamellipodia do not form” and translocation is “severely syndrome patients) compromised” in WASP-deficient cells. See Fig. 5. Badolato et al., 1998 Monocytes (from human Cells from Wiskott-Aldrich syndrome patients remained rounded upon stimulation with chemoattractant, (9670984) Wiskott-Aldrich syndrome and their migration was severely impaired; normal monocytes in their assay showed a polarized actin patients) distribution and readily formed pseudopods. Binks et al., 1998 Dendritic cells (from human Cells from Wiskott-Aldrich syndrome patients were “unable to polarize normally and have severely reduced (9808195) Wiskott-Aldrich syndrome translocational motility in vitro.” (Although they did still see “ruffles” in the WASP-deficient cells, they did patients) not observe larger lamellar structures.) Zicha et al., 1998 Macrophages and Macrophages from Wiskott-Aldrich syndrome patients had defects in directional chemotaxis, although (9674738) neutrophils (from human neutrophils from the same patients were not disrupted. Both mutant and wild-type cells moved at Wiskott-Aldrich syndrome approximately the same speed. Notably, the statistical analysis in this paper eliminated any cell that did patients) not reach a certain distance (60 µm for neutrophils and 10 µm for macrophages), and therefore ignored cells with severely impaired motility. Linder et al., 1999 Macrophages (from human Cells from Wiskott-Aldrich syndrome patients formed fewer podosomes and filopodia, which were (10449748) Wiskott-Aldrich syndrome distributed around the entire cell instead of just at the leading edge. patients) Snapper et al., 2005 Neutrophils (WASP-KO Defect in chemotaxis in WASP-deficient cells (by 25–50%). (15774550) mouse) Kumar et al., 2012 Neutrophils (WASP-KO Cdc42 controls neutrophil chemotaxis and polarity via WASP. “WASP−/− neutrophils have defective (22932798) mouse) chemotaxis and exhibit loss of polarity.” Cells lacking WASP exhibit significantly lower speed (“Sp”) and straightness (“St.”). The authors also report that WASP-deficient cells exhibit a huge increase in the number of protrusions, with many smaller protrusions occurring on the sides of cells instead of at the front. Anderson et al., 2003 Neutrophils (blood from Neutrophils were loaded with purified SCAR or WASP peptides. High concentrations of SCAR severely (12529859) healthy humans) disrupted motility, and WASP had a smaller effect. Zhang et al., 2006 Neutrophils (WASP-KO WASP-deficient cells had impaired adhesion and remain unpolarized, rounded, and without protrusions. (16901726) mouse) Their transendothelial migration was also severely disrupted. Jones et al., 2013 Neutrophils (zebrafish) Inside zebrafish embryos, reduced protrusions and cell velocity in cells with UAS-WASP mutant. See (23868979) Fig. 1 G. Shi et al., 2009 Neutrophils (WASP-KO WASP localizes to pseudopods of chemotacting cells. See Fig. 1 C. (19234535) mouse) Dovas et al., 2009 Macrophages (mouse and Phosphorylation/dephosphorylation of WASP was required for normal podosome formation and turnover as (19808890) human) well as fibronectin matrix degradation. Chemotaxis was impaired by RNAi-reduction of WASP and was rescued by adding WASP, but not by a phosphorylation mutant. Ishihara et al., 2012 Macrophages (WASP-KO WASP is responsible for an initial wave of actin polymerization in response to global stimulation with (22279563) mouse) chemoattractant. Protrusions from WASP-deficient cells were directional, showing intact directional sensing. However, the protrusions from WASP-deficient cells demonstrated reduced persistence compared to wild-type cells. Myers et al., 2005 Dictyostelium Cells with reduced levels of WASP exhibit defects in polarized actin assembly, cell migration, and (15728724) chemotaxis. Veltman et al., 2012 Dictyostelium When SCAR/WAVE is knocked out, WASP assumes the localization and presumably some of the functions (22891261) of SCAR/WAVE. Jain and Thanabalu, Jurkat T cells (human) Knocking down WASP in Jurkat T cells slowed motility and abolished directionality. Overexpression of 2015 (26463123) N-WASP in WASP-KD cells restored the migration velocity without correcting the chemotactic defect. However, insertion of a section of the WASP sequence into N-WASP enabled N-WASP to rescue the chemotactic defect of WASP-KD cells. Worth et al., 2013 Dendritic (WASP-KO mouse) Cells lacking WASP form multiple unpolarized lamellipodia and exhibit migration defects (in persistence (23160469) and directionality, although not speed). Expressing exogenous WASP rescues normal protrusions and migration. Blundell et al., 2008 Dendritic (WASP-KO mouse) Number of podosome protrusions as reduced in WASP-KD cells and could be rescued by transducing with (18388921) WASP. Speed of WASP-KD cells was drastically reduced compared to wild-type and WASP-rescue cells. Zhu et al., 2016 Neuroblasts (C. Elegans) Migrating neuroblasts in developing worms use both WASP and SCAR/WAVE. SCAR mutations reduced (27780040) migration and WASP mutation further impaired motility in SCAR-deficient cells.

Evolutionary roles of WASP and SCAR in motility t'SJU[-BZMJOFUBM 4 Table S2. Cells with adhesion-based motility (expressing N-WASP)

Reference (PMID) Cell type or types Finding

Misra et al., 2007 Adherent fibroblasts (mouse N-WASP deletion disrupts adhesion. (17963692) N-WASPdel/del cell line) Bryce et al., 2005 Adherent fibrosarcoma cells N-WASP KD by siRNA did not reduce lamellipodia formation. By removing an N-WASP activator (16051170) (human) from cells, the lamellipodia were not as persistent, cell migration was defective, and fewer adhesions formed. Desmarais et al., 2009 Carcinoma cells (rat) Cells depleted of N-WASP using siRNA show a defect in in vadopodium-based chemotaxis. (19373774) Sarmiento et al., 2008 Carcinoma cells (rat) siRNA WAVE depletion inhibited lamellipodia formation to a greater degree than N-WASP. (18362183) Depleting both resulted in aberrant jagged protrusion. Benseñor et al., 2007 Slowly crawling MDBK N-WASP, Arp2/3, and actin all localize to protrusions. N-WASP is required for FGF2-stimulated (17264147) (bovine) migration. Lommel et al., 2001 Adherent fibroblasts (mouse Adherent cells lacking N-WASP still form filopodia. (11559594) N-WASPflox/flox) Tang et al., 2013 Carcinoma cells (human A431 “N-WASP has a crucial proinvasive role in driving Arp2/3 complex-mediated actin assembly in (23273897) and HeLa cell lines) cooperation with FAK at invasive cell edges, but WRC depletion can promote 3D cell motility.” Snapper et al., 2001 Fibroblasts (MEFs isolated N-WASP is dispensable for lamellipodia and filopodia formation in fibroblasts. (11584271) from N-WASP+/+ and N-WASPneo/neo) Mizutani et al., 2002 Fibroblasts (rat) N-WASP is essential for podosome adhesion structures and degrading extracellular matrix. (11830518)

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Evolutionary roles of WASP and SCAR in motility t'SJU[-BZMJOFUBM 4